Methods for the treatment of malignant disease in patients using citrulline containing amino acid solutions

ABSTRACT

Disclosed are total parenteral nutrition formulations which include essential amino acids in combination with either arginine or ornithine, for use in the detection of recurrent malignant disease in patients. Such formulations stimulate tumor-specfic polyamine production to a greater extent than non-tumor related polyamine production. Additionally, such formulations were found to specifically promote an increase in red blood cell putrescine levels of tumor-bearing rats. Nontumor-bearing rats were not found to be similarly reactive to these formulations. Methods for making and administering these formulations as well as their use in preventing DFMO-induced toxicity are also disclosed. 
     Also disclosed are parenteral nutritional formulations which include both citrulline and ornithine which have an arginine concentration of less than about 0.10% by weight final concentration. These formulations inhibi 
     The government may own certain rights in the present invention pursuant to NCI grant RQI CA3 4-465.

The government may own certain rights in the present invention pursuantto NCI grant RQI CA3 4-465.

This is a divisional of copending application Ser. No. 07/282,126, filedDec. 9, 1988, U.S. Pat.No. 4,988,724.

BACKGROUND OF THE INVENTION

Reference is made to Applicant's co-pending applications, Ser. No.820,517, filed Jan. 17, 1986 and Ser. No. 002,890, filed Jan. 13, 1987.

FIELD OF THE INVENTION

The present invention relates to improved formulations and methods forthe detection of malignant disease in a patient. More particularly, thepresent invention is directed to formulations which augment tumorrelated increases in a patient's polyamine level and which additionallyprevent drug related side effects in patients undergoing ornithinedecarboxylase inhibitor therapy.

The present invention is also directed to formulations which reducetumor growth rate in a patient.

DESCRIPTION OF THE RELEVANT ART

Polyamines are important in the regulation of protein, RNA and DNAsynthesis in mammalian systems and are essential for cell proliferation.The increased excretion of polyamines in the urine of patients withcancer was reported by Russell, D. H. (1971), Nature, 233: 144-145.Based on animal studies of tumor growth and regression, spontaneouslyand in response to radiation and chemotherapy, a model was proposed byRussell, et al. (1975), Lancet, 2: 797-799, to summarize the potentialrole of polyamines as biochemical markers of human tumor cell growth anddeath. Such observations raise the possibility that measurement ofpolyamine levels in clinical fluids and tissue specimens could be usefulin the diagnosis and elevation of patients with cancer. For our reviewof the diagnostic role of polyamines in cancer, see "Polyamines and theClinical Elevation of Patients with Cancer", Chapter 10, in Progress inCancer Research and Therapy, Raven Press, New York, 1978.

Although polyamine measurements in the urine, plasma and whole blood ofpatients initially held promise as tumor markers, their use fordetecting recurrent systemic disease has been limited. Thisunreliability is due to below clinical sensitivity (high number of falsenegatives) and specificity (high number of false positives). Despitenumerous reports of elevated polyamines in patients with malignantdisease, overlapping standard deviations of polyamine levels betweencancer and non-cancer patients reduce their sensitivity to accuratelydetect malignant disease at an early stage. Furthermore, increasedpolyamine levels have been associated with non-malignant disease statessuch as certain inflammatory and infectious diseases. Such unreliabilitycould be removed by the availability of techniques for specificallyincreasing polyamine levels observed in false positives such asinfectious disease patients. Accordingly, techniques for improving thesensitivity and specificity of the polyamine level test as an indicatorof malignant disease in man would represent a significant advance inmedical science.

Nutritional status plays an important role in the clinical management ofpatients with malignant disease. Warren, S. (1932) Am. J. Med. Sci.:184: 610, and, more recently, others (DeWys, W. D., Begg. C., Lavin, P.T., et al. (1980) Am. J. Med Sci.: 69: 491-497) have shown thatmalnutrition can significantly influence the mortality and shorten thesurvival time of patients with disseminated disease. Furthermore, cancertherapy may be delayed in patients with a compromised nutritional statusfor fear of reducing oral intake. In 1967, Dudrick, et al. (1968)Surgery, 64: (1969) 134, Ann. Surg., 169: 974 introduced a safe andeffective method to parenterally feed patients until gastrointestinalfunction could be restored. Soon, total parenteral nutrition (TPN) wasused for patients in many diseased states such as congenital anomaliesof the gastrointestinal tract, trauma, inflammatory bowel disease, andliver disease. Fischer, U.S. Pat. No. 3,950,529 (1976).

Copeland and Dudrick, (1976) Curr. Probl. Cancer, 1: 3, recognized thepotential of parenteral feeding techniques to solve nutritional problemsin a cancer population. It was then theorized that if nutritionaltherapy in conjunction with cancer therapy could improve hostnutritional status, then host survival should improve. Also, preliminaryuncontrolled studies showed that host tolerance and tumor response toantineoplastic therapy improved when malnourished patients received TPN,advancing the theory that force feeding of nutrients reduced hosttoxicity and sensitized tumors to chemotherapeutic agents (Isell, B. F.,Valdivieso, M., Zaren, H. A., et al. (1978) Cancer Treat Rep., 62: 1139;Lanzotti, V. J., Copeland, E. M. III, George, S. L., et al. (1975)Cancer Treat Rep., 59: 437).

In the 1970's, several prospective randomized trials were conducted totest these theories. These trials involved patients requiring systemicchemotherapy for testicular carcinoma, small cell lung carcinoma,lymphoma, lung adeno carcinoma and colorectal carcinoma (Lanzotti, etal., (1975) Cancer Treat. Rep., 59: 437; Samuels, M. L., et al. (1981)Cancer Treat, Rep., 65: 615-627; Popp, M. D., et al., (1981) CancerTreat. Rep., 65: 129-135; Valdivieso, M., et al., (1981) Cancer TreatRep., 659 (suppl. 5): 145; Nixon, D. W., et al., (1981) Cancer Treat.Rep., 65 (suppl.

5): 121-128; Jordan, W. M., et al., (1987) Cancer Treat. Rep., 65: 197.The results of these trials showed that TPN did not improve hosttolerance to chemotherapy. Furthermore, tumor response rates wereindependent of host nutritional status. In fact, one report suggeststhat TPN may have led to earlier patient demise secondary to progressivedisease. Nixon, D. W., et al., (1981), Cancer Treat. Rep., 65 (suppl.5): 121-128).

Several early studies in animal-tumor models in the late 1970's andearly in 1980 showed that nutritional therapy (TPN) can result in tumorgrowth and, in some cases, accelerate growth. Cameron, I. L., (1981),Cancer Treat. Rep.. 65 (suppl. 5): 93; Buzby, G. P., Mullen, J. L.,Stein, T. P., et al., (1980), Cancer 45: 2940. For some tumors, tumorgrowth can also occur during host starvation, emphasizing that tumorswill obtain their exogenous or endogenous nutrients for their energyneeds and biosynthetic pathways, to the host's demise. (Sauer, L. A.,Nagel, W. O. Dauchy, R. T., et al., (1986) Cancer Res., 46: 3469).Therefore, the use of a standard TPN formulation was contraindicated forpatients with malignant disease.

These preliminary studies, however, did not consider significantbiological aspects of host-tumor metabolism, namely the competition ofhost and tumor tissues for nutrients in devising an appropriate TPNregiment. Accordingly, a formulation which would provide nutritionalsupport to the patient with malignant disease without stimulating tumorgrowth would significantly advance the currently practiced clinicaloncological management of these patients.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide an amino acidformulation for improving the diagnostic reliability of polyamine leveldeterminations as indicators of malignant disease in man. In particular,amino acid formulations prepared in accordance with the presentinvention and administered to patients promote a specific elevation inthe polyamine level of patients with primary or recurrent malignantdisease. The term amino acid, as used herein, is defined to include allamino acid-derived compounds which will effectively provide the free1-amino acid at the intracellular level. For example, peptides are aminoacid-derived compounds that when acted upon by metabolic enzymes willprovide the free amino acid. Similarly, amino acid derivatives, forexample, n-acetylated amino acids, are included within the meaning ofthe term amino acid.

Formulations include a pharmacologically acceptable amount of apolyamine precursor in combination with at least one of the amino acids,preferably one of the essential amino acids. Essential amino acids asused herein refer to those found by William Rose and his associates tobe essential in that they are not normally metabolically produced by thebody. For example, see Rose (1949) Fed. Proc., 8:546 and Rose et al.(1955) J. Biol. Chem., 217:987. Additionally, as used herein, polyamineprecursors are defined as those amino acids in the metabolic pathway ofurea and polyamine production and include methionine, arginine,ornithine and citrulline.

Such formulations may include any non-toxic amount of each of theincluded amino acids. Therefore, for the purposes of the presentinvention, a pharmacologically acceptable amount of an included aminoacid generally means any level of that amino acid which is at least highenough to supply minimal metabolically sufficient concentrations of thatamino acid to the target cells. However, such formulations couldpresumably incorporate amino acid concentrations which approachtoxicity-inducing levels, for example, in an attempt to "tailor"formulations to fit a particular patient population. In a preferredembodiment, the concentration of included amino acids found to promotegood polyamine responses exhibited a range from 50 to 200 mg. fortryptophan to 250 to 1400 milligrams for leucine in every 100milliliters of a parenteral formulation. The preferred range forarginine, ornithine or methionine, the polyamine precursors, is between50 and 750 mg/100 ml. of solution, however, higher concentrations may beemployed where desired. In a more preferred embodiment, theconcentration of polyamine precursor is approximately 30 mM (which, forexample, corresponds to 450 mg. ornithine/100 ml. of the formulation).

In another embodiment, the amino acid formulation is convenientlysupplied by combining pharmacologically acceptable amounts of ornithineor arginine with commercially available parenteral feeding solutions.Such feeder solutions could prove useful both as parenteral solutionstailored to maximize the sensitivity of detection of recurrent malignantdisease and as adjuvants to the cancer-screening of patients who areundergoing parenteral feeding.

It is a further object of the present invention to provide methods fordetecting cancer in a patient which generally includes administering tothe patient one of the parenteral amino acid formulations described anddetecting an increase in the patient's polyamine level followingadministration of the formulation, a relative increase being indicativeof cancer in the patient. Relative increase in polyamine levels is meantto include either relative increases with respect to the cancerpatient's own pre-administration level or with respect to "normal"patient population's average pre- or post administration polyaminelevels.

Although the full advantages of the present invention are particularlyexemplified through detection of a relative increase in putrescine,measurement of all of the polyamines, including spermidine, spermine andputrescine, is sufficient for most uses.

Similarly, although polyamine levels may be determined in any of anumber of patient samples, including cerebrospinal fluid, urine, plasma,serum and whole blood, 20 in a preferred embodiment, the patient's redblood cell (RBC) polyamine levels are measured as a more sensitiveindicator of polyamine levels.

It is still further object of the present invention to provideformulations for use in preventing or reducing the occurrence ofODC-related toxicities in patients undergoing ODC therapy. In thisregard, it is demonstrated herein that amino acid formulations which areformulated to include a polyamine precursor, preferably ornithine, canreduce or reverse DFMO induced thrombocytopenia. Moreover, it has beenfound that for such uses, polyamine precursors may be administeredeither alone or in combination with other amino acids.

It is still a further feature of the present invention to provideformulations that reduce*I903* tumor growth rate in patients withmalignant disease. In this regard, it is demonstrated herein that aminoacid formulations which are formulated to decrease or eliminate arginineand which include ornithine can reduce the rate of TPN induced tumorgrowth. It is further proposed that a formulation which includescitrulline can be used in conjunction with or instead of ornithine tofurnish necessary urea cycle substrates. In one preferred embodiment,the final concentration of included arginine is less than about 0.10% byweight where citrulline is present in a non-toxic pharmacologicallyacceptable concentration. A preferred embodiment of this particularformulation includes citrulline in about a range of 0.01 to 2% by weightfinal concentration. In another preferred embodiment, the concentrationof included arginine was less than about 0.05% by weight of theformulation where ornithine and citrulline are present in a non-toxicpharmacologically acceptable concentration. A preferred embodiment ofthis particular formulation includes ornithine in a greater than 0.5% byweight final concentration and citrulline in a range of 0.01 to 2% byweight final concentration of the formulation.

Another embodiment of the formula does not include citrulline, but doesinclude arginine in a less than about 0.10% by weight finalconcentration and ornithine in a non-toxic pharmacologically acceptableconcentration. A preferred embodiment of this particular formulationincludes ornithine in about a 1% by weight final concentration. However,it is postulated that the lower the concentration of arginine in each ofthe above formulations, the greater the tumor-inhibiting effect will be.

In an additional embodiment, the formulation further includes a mixtureof essential amino acids and at least one non-essential amino acid. Thenon-essential amino acid of choice in one such preferred embodiment isalanine, to be included in any non-toxic, pharmacologically acceptableconcentration. One particular embodiment of the present inventionincludes alanine in about a 1% by weight final concentration. Theseparticular preferred embodiments are arginine-free. Such formulationsmay include any non-toxic, pharmacologically acceptable concentration ofeach of the included essential amino acids.

An additional preferred embodiment of the formulation includes a mixtureof essential amino acids in pharmacologically acceptable concentrations,less than about 0.10% arginine by weight final concentration , bothornithine and citrulline at a non-toxic, pharmacologically acceptableconcentration, and a mixture of the non-essential amino acids: alanine(200-3500 mg./100 ml); glycine (250-3000 mg./100 ml); proline (100-1500mg./100 ml) and serine (5-650 mg./100 ml). A pharmacologicallyacceptable amount of an included amino acid in any of the aboveformulations generally means any level of that amino acid which is atleast high enough to supply minimal metabolically sufficientconcentrations of that amino acid to the target in the feeding solutioncells, but not so high as to be toxic. For example, concentrations inthe range of about 0.01% and 5.0% are for most amino acids considerednon-toxic and pharmacologically acceptable. This range applies as wellto citrulline and ornithine. Also, it is observed that the lower theconcentration of arginine in each of the above formulations, the greaterthe tumor-inhibiting effect will be. In fact, a less than about 0.10% byweight final weight concentration of arginine is hypothesized to have agreater tumor growth inhibiting effect. A formulation without argininewould provide the most superior tumor growth inhibiting effect of allthe presently disclosed formulations.

It is a further object of the present invention to provide methods forinhibiting or eliminating tumor growth in a patient with malignantdisease, which generally includes administering to the patient one ofthe amino acid formulations described herein. The mode of administeringsaid formulation is parenterally, however the formulas could be modifiedso as to allow oral administration.

Successful inhibition of tumor growth may be detected throughdemonstration of relatively increased survival rates offormulation-receiving patients compared to standard TPN receivingpatients. Survival data taken from patients undergoing chemotherapy andcancer patients being prepared for surgery suggest that standard TPNsupport solutions may actually decrease survival rate. However,nutritional support is often times indicated in persons undergoingchemotherapy, as intestinal complications accompanying such treatmentmake oral feeding difficult. Patients with acute disruption ofgastrointestinal functions and those with such disorders awaiting majorsurgery frequently suffer from a compromised nutritional status.Correction of varying states of malnutrition with a tailored amino acidregimen is hypothesized to constitute both a life-sustaining tacticuntil intestinal function returns to normal and a beneficialpre-operative practice. A decrease in postoperative complications withshorter recovery times are particular expected benefits of such apre-operative practice. The relative nutritional status of a patient maybe determined by numerous methods well know to those of skill in theart, including measurement of plasma proteins, anthropometricmeasurements, immunological testing, urine chemistries and bodycompositional studies.

An additional object of the present invention is to provide a processfor preparing a formulation that decreases tumor growth rate. In itsbroadest embodiment, this process comprises the steps of combiningnon-toxic, pharmacologically acceptable amounts of citrulline witharginine, and adding to this combination a sufficient volume ofappropriate liquid medium so as to achieve about a pharmacologicallyacceptable concentration of citrulline and a less than 0.10% by weightconcentration of arginine. In a preferred embodiment, a glucose solutionor filtered water is an appropriate liquid medium. It has been foundthat for such uses, this solution may be administered in combinationwith other amino acids and ornithine.

BRIEF DESCRIPTION OF THE DRAWINGS

Table 1. A listing of several parenteral formulations, and theirmanufacturers, which include the indicated amount of amino acids perevery 100 ml. of the formulation.

FIG. 1. Graphs demonstrate changes in RBC putrescine, spermidine, andspermine levels in non-cancer patients and patients with clinicallyoccult malignant disease during TPN (total parenteral nutrition)therapy. PRE indicates RBC polyamine levels before preoperative TPN wasstarted, and POST indicates levels after 7-10 days of preoperative TPNbut before surgery. N.S. indicates not significant.

FIG. 2. Graphs demonstrate changes in RBC putrescine: spermidine andspermine: spermine ratios in non-cancer patients and patients withclinically occult malignant disease during TPN. PRE indicates RBCpolyamine ratios before preoperative TPN was started and POST indicatesratios after 7-10 days of preoperative TPN but before surgery. N. S.indicates not significant.

FIG. 3. Platelet counts plotted against dose of DFMO given as acontinuous i.v. infusion for 12 days in tumor-bearing (O) andnon-tumor-bearing rats (o). Bars indicate standard deviation. As thedose of DFMO increases, there is significant platelet suppression. Thisexperiment was done to determine if simultaneous ornithine infusioncould block platelet suppression.

FIG. 4. Platelet counts plotted against days of continuous i.v. DFMOinfusion in patients with advanced colorectal carcinoma. The upper graphshows the platelet suppression at 6 g DFMO/sq m/d. The lower graphdepicts the platelet suppression at 8 g DFMO/sq m/d. Each course ofinfusion is shown by two points representing day 1 of infusion and day22 to 28 when DFMO was discontinued. The paired points are connected bya straight line. The data show that platelet suppression is the majorlimiting host toxicity of DFMO given as a continuous infusion. Thesimultaneous infusion of ornithine with DMFO may inhibit platelettoxicity.

FIG. 5. TPN-induced tumor growth during the plateau phase of coloncarcinoma. Fisher 344 rats with 3 g transplantable colon tumors growings.c. in the R flank were randomized to 2 groups and a central venouscatheter was inserted. CHOW received i.v. saline and oral chow adlibitum for 12 days. TPN consisted of 500 ml D50W+500 ml 10% Travasol(general amino acid solution) and was administered for 12 days to theother group. Note that TPN was administered during the period of timewhen the tumor was entering the plateau phase of growth. The final tumorweight (after 12 days TPN) for the TPN group was significantly greaterthan the CHOW final tumor weight (P<0.05). a=P<0.05 compared with CHOWfinal tumor weight.

FIG. 6(a) and (b). Graphs demonstrate the Gompertzian growth curve ofthe methylcholanthrene-induced sarcoma (MCA) and the Ward coloncarcinoma (WCC) in the Fisher 344 rat. The MCA reaches its growthplateau at 60 grams while WCC reaches its plateau at 9 grams. Theplateau phase of growth is related to the solid tumor outgrowing itsblood supply and, hence, the reduced availability of O₂ and nutrients.There is no change in tumor growth when TPN is administered during thelog phase of MCA growth. Host and MCA growth are significantly reducedwhen the host is fed a restricted diet. However, MCA growth becomesexponential when TPN is administered after 14 days of restricted diet.

FIG. 7. Graph demonstrates the role of arginine in TPN (total parenteralnutrition) induced tumor growth observed in Example VIII. Fisher 344rats with approximately 3 g transplantable colon tumors growing in the Rflank were randomized to four groups and a central venous catheter wasinserted. CONTROL received i.v. saline and oral chow ad libitum. Thedaily intake for these rats was 52±9 cal and 584±104 mg nitrogen. TPNconsisted of 500 ml D50+500 ml 10% Travasol. The daily intake of TPN was54±2 cal and 550±15 mg nitrogen. EAA describes a parenteral regimenconsisting of 500 ml D50+500 ml 5.2% Aminess (essential amino acidonly). The daily intake of EAA formula was 5±4 cal and 270±10 mgnitrogen. EAA+NEAA describes a parenteral regimen consisting of 500 mlD50+500 ml 5.2% Aminess with non-essential amino acids except arginine.The daily intake of EAA+NEAA formula was 51±5 cal and 574±15 mgnitrogen. Hence, TPN and EAA±NEAA regimens were isonitrogenous andisocaloric with the only difference being the content of arginine. Theparenteral regimens and saline were administered by continuous i.v.infusion for 12 days. This figure shows that TPN resulted in increasedtumor growth compared with CONTROL. When EAA was given, no tumor growthwas seen. When EAA+NEAA was given, there was a slight increase in tumorweight but much less than TPN.

This study suggests that arginine may be important in stimulating tumorgrowth. Bars indicate standard deviation. Numbers indicate number ofanimals. a=P<0.05 vs CONTROL; b=P<0.05 vs TPN; c=P<0.05 vs EAA.

FIG. 8. Graph demonstrates the role of polyamine precursors inTPN-induced tumor growth observed in Example IX. Approximately 4 g. Wardcolon carcinomas were established in Fisher 344 rats that receivedparenteral therapy. Rats were then randomized to four treatment groupsCONTROL (i.v. saline+chow), ALANINE, [(500 ml D50 +250 ml. 5.2%EAA+alanine (3.3 mmol/100 ml)], ARGININE [(500 ml D50+250 ml. 5.2%EAA+arginine (3.3 mmol/100ml), and ORNITHINE [(500 ml D50+250 ml. 5.2%EAA +ornithine. (See Table IX). The regimens were administered bycontinuous i.v. infusion for 6 days. The results show the percentincrease in tumor weight during the 6-day infusion with the barsindicating the standard deviation. The numbers indicate number ofrats. * indicates P<0.05 compared with the ARGININE group. CONTROL andORNITHINE were not significantly different. The data indicate that thearginine in TPN solutions may stimulate tumor growth while ornithinedoes not.

FIG. 9. Graph demonstrates the effect of decreasing arginineconcentrations on tumor growth measured in grams. As shown, arginineconcentrations as low as 0.1% arginine in the TPN solution may increasetumor growth.

FIG. 10. Graph demonstrates the effect of decreasing arginineconcentrations on urine ammonia determinations. The saline group had nodetectable ammonia in the urine. The reduction of arginine in the TPNsolution from 0.65 to 0.1 g./100 ml. resulted in increasing ammonialevels in the urine. Plasma ammonia levels were the same for all groups(data not shown).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention, in its most general and overall scope, isdirected to amino acid formulations, including amino acid formulationswhich contain the amino acid ornithine, methionine or arginine, usefulin the stimulation of polyamine formation in the tumor-bearingindividual. Methods are provided which utilize these amino acidformulations to improve the diagnostic reliability of polyamine leveldetermination in cancer screening and cancer staging. Additionally, suchformulations provide the capability of reducing toxicities associatedwith anti-tumor chemotherapy aimed at blocking polyamine production.

The present invention is also directed to an amino acid formulation forpatients suffering from malignant disease. Additionally, methods areprovided which utilize these amino acid formulations to reduce tumorgrowth rate. A process for preparing the subject amino acid formulationsis also revealed.

a. INTRODUCTION

Polyamine production is associated with cell division. Increases in theintracellular levels of polyamines, particularly putrescine andspermidine, in the early phase of both normal and neoplastic cellproliferation are well documented. Conversely, a reduction in tumorburden is associated with a reduction of polyamine levels suggesting thepotential use of polyamines as a biological marker of tumor growth. Asubstantial portion of circulating polyamines are carried in the RBC.Because the enzyme systems necessary to synthesize polyamines are notfound in enucleated RBC, it is theorized that RBC are carriers ofpolyamines from sites of production to sites of conjugation andexcretion.

The pathway for polyamine synthesis begins with L-ornithine. Thisnatural amino acid, although not normally incorporated into proteins, ispart of the urea cycle which metabolizes arginine to ornithine andureas. Ornithine is converted by ornithine decarboxylase (ODC) toputrescine and CO₂, the rate-limiting step in the production ofpolyamines. Putrescine is converted to spermidine by spermidinesynthetase in association with the decarboxylation ofS-adenosylmethionine by S-adenosylmethionine decarboxylase. Spermidineis then converted to spermine by spermine synthetase, again inassociation with the decarboxylation of S-adenosylmethionine.Putrescine, spermidine and spermine represent the three primarypolyamines.

It has been found that infusion of feeding solutions which containprecursors of polyamine metabolism, alter polyamine metabolism in thetumor-bearing host. One example is parenteral nutrition formulationswhich contain methionine, arginine and/or ornithine. Total parenteralnutrition formulations (TPN) are specific feeding solutions whichgenerally contain higher amino acid concentrations than supplementaryfeeding solutions, for example. TPN is a technique used to intravenouslyfeed malnourished cancer patients. These nutrient solutions generallycontain concentrated glucose, crystalline amino acids, electrolytes andvitamins. The amino acid compositions of typical commercially availableamino acid solutions are shown in Table 1. Each amino acid is purchasedin crystalline form and compounded into amino acid solutions. Uniqueamino acid solutions are marketed for patients with kidney failure,liver failure and trauma patients. However, no special solutions yetexist for cancer patients.

Studies have shown that the polyamine biosynthetic pathways may beimportant in the development of amino acid solutions specific for cancerpatients. An important consideration is the interaction between tumorpolyamine production and a specific amino acid solution that may enhanceRBC polyamines as tumor markers. It has been determined thatadministration of TPN solutions to patients with occult malignanciesproduce changes in polyamine levels compared with non-cancer patientswhile plasma CEA and creatine kinase-brain band levels did not change,indicating a specific increase in polyamine levels in tumor-bearingpatients. Accordingly, the sensitivity and specificity of RBC polyaminemeasurements to detect occult disease may be enhanced with new aminoacid solutions which include substrates for tumor polyamine productionsuch as ornithine or arginine.

An additional important consideration in polyamine metabolism is theinteraction between tumor polyamine utilization and specific amino acidsolutions. It has been determined that administration of standard TPNsolutions, which typically contain arginine, actually increase the rateof tumor growth while formulations with decreased standard arginineconcentrations result in decreased tumor growth. It is hypothesized thisdata indicates arginine may act to promote tumor growth. Accordingly,the growth rate of tumors may be reduced with new amino acid solutionswhich decrease or eliminate this arginine substrate hypothesized to beutilized in tumor polyamine metabolism.

In recent years, chemotherapeutic agents that directly inhibit polyaminesynthesis have been developed. Difluoromethylornithine (DFMO), one suchdrug, is an irreversible inhibitor of ODC and potentially can be givencontinuously with significant anti-tumor effects. This drug isrelatively non-toxic to the host while producing inhibition ofputrescine synthesis in tumors. Studies in a rat-tumor model demonstratethat DFMO infusion can produce a 90% decrease in tumor putrescine levelswithout suppressing peripheral platelet counts.

Although DFMO can effectively block tumor putrescine biosynthesis, theresultant anti-tumor effect is cytostasis and not cytotoxicity. Forexample, DFMO reduces the growth rate of an MCA sarcoma but does notproduce tumor regression. This finding is consistent with reports ofother investigators who showed that DFMO is a cytostatic agent. However,studies indicate that a significant role exists for DFMO agents,permitting the future development of combination chemotherapeuticregimens which incorporate DFMO.

Although the toxicity associated with DFMO therapy is not, in general,as severe as other types of chemotherapy, in limited clinical trials ithas been found to promote a dose-related thrombocytopenia. Moreover,studies in rats have shown that continuous infusion of DFMO for 12 dayssignificantly reduces platelet counts compared with controls. Otherinvestigations have made similar observations in which thrombocytopeniais the major toxicity of continuous i.v. DFMO therapy. These findingssuggest that DFMO may significantly inhibit ODC activity of the bonemarrow precursors of megakaryocytes. It is contemplated by the presentinventors that the formulations disclosed herein, in addition topromoting tumor polyamine production for diagnostic purposes, constitutespecific antidotes to ODC-directed chemotherapy.

One means to reverse DFMO toxicity in the host is to infuse amino acidprecursors of polyamine biosynthesis. Special amino acid solutions richin ornithine or arginine made in accordance with the present inventioncan rescue the bone marrow from the toxic side effects of DFMO. Forexample, the addition of ornithine to feeder solutions in a rat-tumormodel resulted in a significant reversal in the platelet suppression.Additionally, the constant infusion DFMO technology in a rat-tumor modelpermits the ability to test such new amino acid solutions in reducingDFMO induced thrombocytopenia. It is hypothesized that such solutionsprovide substrates which stimulate the production of polyamines innormal cells which are sensitive to ODC-directed chemotherapy, thuspromoting recovery from toxic side effects of the drugs. Similarly, itis hypothesized that solutions containing polyamine precursors may serveto enhance ODC-directed chemotherapy against certain tumors, as adjuantsto radiation therapy or may in themselves exhibit anti-tumor activityagainst some tumors.

Solutions prepared with essential amino acids+ ornithine produce higherRBC putrescine levels compared with essential amino acids or essentialamino acids+arginine. RBC and tumor spermidine and spermine levels arenot affected to as great an extent by these solutions. The specificincreases in putrescine levels may be explained by the proximity ofcertain amino acid components to the pathways of polyamine synthesis.Use of these new amino acid solutions could be important to detectoccult malignancy following surgical resection. For example, colorectalcarcinoma patients represent an ideal population to test thetumor-detecting capabilities of RBC polyamine levels enhanced by aminoacid solutions. Such detection studies could be done, for example, twoto three months postoperatively.

Use of a new amino acid solution to enhance the cancer detectingproperties of blood polyamine measurements represents an improvement ofcurrently available diagnostic methods. Breast and colorectal cancersrepresent possible malignancies where this may be applicable. Pathologicassessment of axillary lymph node involvement is the only means topredict risk of future metastatic disease in women with either clinicalstage I or II disease. By increasing our ability to select patients withpositive lymph node involvement, the benefits of adjuvant chemotherapywill be increased. Similar application can be made for patients withDukes B₂ or C₂ rectal carcinomas. CEA measurements or diagnosticradiologic tests are not effective for selecting those patients withmicroscopic residual disease that can be treated by postoperativeradiotherapy to the pelvis. Amino acid solutions with a novel amino acidsuch as ornithine could enhance polyamine detection of residual diseaseand, thereby, select patients who would benefit the most frompostoperative adjuvant radiotherapy.

Enhanced cancer detection with polyamine measurements after TPNadministration potentially represents considerable improvement overother tumor markers. Infusion of amino acid solutions rich in ornithineincrease tumor polyamine production, thus elevating their levels inperipheral blood. Infusion of arginine or ornithine alone, that is, notin combination with one or all of the essential amino acids, has notproduced elevated blood polyamine levels in the experimental tumorsystem.

b. DETERMINATION OF POLYAMINE LEVELS

Numerous techniques are known in the art for determining polyaminelevels in aqueous biological samples such as urine and plasma.Generally, such techniques involve subjecting the aqueous sample toamino acid analysis by an automated amino acid analysis. In this manner,individual determinations of putrescine, spermidine and spermine may bemade. More recently, enzymatic methods have been developed as disclosedin Japanese patents 8402700 and 8482099.

Although urine and plasma may be utilized for polyamine determinationsin accordance with the present invention, it has been determined by thepresent inventors that red blood cell polyamine levels more accuratelyreflect tumor-related increases in polyamine production. Therefore,determination of RBC polyamines is a preferred method where accuracy isrequired, although this procedure is somewhat more involved. Thefollowing method is a representative RBC polyamine level determination.

After obtaining the blood sample by venipuncture, or some other suitablemeans, blood hematocrit is determined and the blood is centrifuged for10 minutes at 500 g. The plasma is removed for albumin determination andthe remaining cells are washed with an equal volume of cold 0.9% NaCland centrifuged at 200 g for 15 minutes. The buffy coat is thencarefully aspirated along with the supernatant and discarded. Afterthoroughly mixing the packed RBC, a 1.0 ml aliquot is extracted forpolyamine analysis. While shaking continuously with a Vortex mixer, 2.5ml of freshly prepared 6% trichloroacetic acid (TCA) is added followedby 60 ul of 100% TCA. This is done to avoid insufficient mixing if 100%TCA is added directly to RBC. The samples are mixed thoroughly for aminimum of 3 minutes and frozen at -70° C. if further extraction stepsare performed. The samples are thawed and centrifuged at 200×g for 15minutes. The supernatant is then transferred to tubes containing 40 ulof concentrated HCl. The samples are washed twice with anhydrous etherand dried. The pellets are dissolved in 100 ul of 0.5 N HCl andcentrifuged at 200 g. The clear supernatant is then analyzed forpolyamines using, for example, a Durrum D-500 amino acid analyzer.

In an additional embodiment, the patient's RBC polyamine level isdetermined both before and after administration of amino acid solutions.This allows the physician to both identify the "reactivity" of thepatient's polyamine level and to further compare with normal profiles.The following example demonstrates the use of total parenteral nutritionsolutions to detect recurrent malignant disease.

EXAMPLE I: DETECTION OF RECURRENT MALIGNANT DISEASE BY POLYAMINEANALYSIS IN PATIENTS RECEIVING TOTAL PARENTERAL NUTRITION

This study was performed to demonstrate that total parenteral nutrition(TPN) results in significant increases in erythrocyte (RBC) polyaminelevels in patients with clinically occult recurrent malignant disease.TPN was administered preoperatively to six noncancer patients and sevenpatients who had a history of curative operation for malignant diseaseand, after receiving TPN, were found to have recurrent disease. RBCputrescine (PUT), spermidine (SPD), and spermine (SPM) were determinedbefore and after preoperative TPN in each patient. Plasmacarcinoembryonic antigen (CEA) and creatine kinase brain band (CK-BB)were also measured during the study as controls. Mean length (±S.E.) ofpreoperative TPN for the noncancer group and the group harboring occultrecurrent disease was 8.4±2.9 days and 9.6±3.6 days, respectively, Therewere no significant changes in RBC polyamine and plasma CEA and CK-BBlevels in the noncancer group after TPN. Those patients with occultmalignant disease had significant increases in RBC PUT and PUT/SPD ratio(p .05) during TPN, while RBC SPD and SPM and plasma CEA and CK-BB didnot change. The data indicates that short-term TPN can enhance the useof polyamine measurements as markers of recurrent malignant disease.

PATIENT POPULATION

Patients who required preoperative TPN in order to restore or preventnutritional deficits before an operative procedure were entered intothis study. Patients who received chemotherapy or radiation therapy orunderwent an operative procedure within three months of this study wereexcluded. Women with active ovulatory cycles were also excluded. Theonly treatment given during preoperative TPN was nutritional therapy.All patients had normal renal function (plasma creatinine less than 1.4mg/dL and BUN values less than 25 mg/dL) and normal liver function(serum bilirubin less than 1.4 mg/dL) as prerequisites for entry intothe study. This study was conducted with the approval of the HumanSurveillance Committee of M. D. Anderson Hospital and the informedconsent of the patient.

There were six patients who required preoperative TPN and who were freeof malignant disease, as documented by exploratory laparotomy,histologic evaluation of the respected specimens, and length ofdisease-free status (greater than five years). There were seven patientswith clinically occult recurrent malignant disease during their courseof preoperative TPN. These patients had had either a "curative"operative procedure or had a suspicious lesion that could not be provenas recurrent malignant disease by diagnostic evaluation, which includedroentgenographic and endoscopic studies. Disease status was determinedby exploratory laparotomy following preoperative TPN or by follow-upclinic visits.

NUTRITIONAL REGIMEN

TPN solutions consisted of 50% glucose (500 ml), 10% crystalline aminoacids (500 ml), NaCl (40-60 mEq/L), KCl (20-40 mEq/L), KH₂ PO₄ (10-15mEq/L), Ca gluconate (4.5 mEq/L), MgSO₄ (10-15 mEq/L), MVI-12 (10 ml),and trace elements (2 ml). A 10% soybean oil emulsion (500 ml) wasadministered biweekly. TPN solutions were administered continuouslythrough a central venous catheter at a rate of 30-50 calories/kg bodyweight/day and 2.0-3.0 g amino acid/kg body weight/day. Fluid andelectrolyte balance was monitored every Monday, Wednesday, and Friday.

STUDY DESIGN

Each patient was hospitalized for 7 to 10 days of preoperative TPN.Before TPN was started, venous blood (7 ml in a heparin-coated tube) wasobtained. After completion of preoperative TPN and on the day before theoperative procedure, a second venous blood sample was obtained. RBCpolyamine determinations and plasma CEA and CKBB measurements were donebefore and after preoperative TPN for each patient. In this manner, eachpatient served as his or her own control in determining the effect ofTPN on RBC polyamines and plasma CEA and CK-BB levels. Analysis of datawas done by a paired Student's t-test, comparing levels before and afterTPN within the noncancer and cancer groups.

POLYAMINE, CEA AND CK-BB DETERMINATIONS

RBC putrescine, spermidine, and spermine levels were measured by highperformance liquid chromatography using a Durrum D-500 amino acidanalyzer, (Dionex Corp., Palo Alto, Calif.) as described above. PlasmaCEA was determined by an enzyme immunoassay procedure kit (AbbottLaboratories, North Chicago, ILL) and plasma CK-BB was measured with aradioimmunoassay method (Mallinkrodt, Inc., St. Louis, Mo.).

The clinical history of each patient is shown in Table II. The sixnoncancer patients received preoperative TPN for 8.4±2.9 days(mean±S.E.). Patients #4 and #5 were both studied twice, six and twelvemonths apart, because of two separate episodes of weight loss induced byshort gut syndrome. This accounts for the eight determinations ofsequential polyamine measurements for this group. There were sevenpatients with clinically occult malignant disease. Three patients hadrecurrent disease at laparotomy immediately following preoperative TPN.The other four patients developed recurrent disease at three months totwo years after the study. The mean length of TPN for this group was9.6±3.6 days. Mean weight loss, based on usual body weight, was 7.1±1.1%TPN was started. Preoperative TPN was given without infectious ormetabolic complications.

                  TABLE II                                                        ______________________________________                                        Patient Population                                                            Age   Sex     Diagnosis                                                       ______________________________________                                        Patients with Occult Malignant Disease                                        59    F       Recurrent bladder carcinoma 9 mos.                                            after TPN                                                       58    F       Recurrent esophageal carcinoma 3 mos.                                         after TPN                                                       49    M       Recurrent pancreatic carcinoma 7 mos.                                         after TPN                                                       60    M       Recurrent esophageal carcinoma discovered                                     at laparotomy                                                   40    M       Recurrent esophageal carcinoma discovered                                     at laparotomy                                                   48    F       Gastric carcinoma discovered at laparotomy                      78    F       Recurrent colon carcinoma 24 mos. after TPN                     Patients with Benign Disorders                                                50    F       Benign esophageal stricture                                     55    F       Benign gastric ulcer                                            30    F       Pseudointestinal obstruction                                    20    F       Chronic radiation enteritis and short gut                                     syndrome                                                        51    M       Chronic radiation enteritis and short gut                                     syndrome                                                        71    F       Benign gastric ulcer                                            ______________________________________                                         *patients were studied twice for a total of eight sequential polyamine        studies for the noncancer group.                                         

Polyamines could not be detected in TPN solutions. Changes in RBC count(×10⁶ /mm³) were similar for both groups with a mean decrease of 4.3%during TPN. FIG. 1 shows the changes in RBC putrescine, spermidine, andspermine for both groups. Eight determinations in six noncancer patientsshowed no significant increases in RBC polyamine levels during TPN. Thepatients with clinically occult malignant disease had a significantincrease in RBC putrescine levels (p<0.01), while RBC spermine levelsincreased but not significantly. There was a trend toward higher, butnot significant, spermidine levels in this group (p<0.07).

Changes in RBC putrescine: spermidine and spermidine: spermine ratiosduring TPN for both groups are shown in FIG. 2. The spermidine: spermineratio did not change for either group. The patients with clinicallyoccult malignant disease had a significant increase in the putrescine:spermidine ration, while the noncancer group did not. Plasma CEA andCK-BB levels did not increase significantly during TPN in either groupof patients as shown in Table III.

                  TABLE III                                                       ______________________________________                                        Plasma CEA and CKBB Data During TPN.sup.a                                             Plasma CEA (ng/ml).sup.b                                                                   Plasma CKBB (ng/ml).sup.b                                n         Pre       Post     Pre     Post                                     ______________________________________                                        Noncancer                                                                             6     2.9 ± 1.6                                                                            2.8 ± 1.4                                                                         3.6 ± 2.1                                                                          3.5 ± 2.4                           Occult                                                                        Cancer  4     2.4 ± 2.0                                                                            5.0 ± 4.9                                                                         1.9 ± 0.5                                                                          1.6 ± 0.3                           ______________________________________                                         .sup.a Plasma carcinoembryonic antigen (CEA) and creatine kinase brain        band (CKBB) were determined before (Pre) and after (Post) preoperative TP     in noncancer patients and patients with clinically occult malignant           disease. Normal values for CEA are 0-3 ng/ml (nonsmokers) and 0-6 ng/ml       (Smokers).                                                                    .sup.b Values represent ± SD                                          

The previous example demonstrates that two procedures may enhance thevalue of polyamines as markers of recurrent malignant disease. First,using each patient as his or her own control, polyamine measurementswere determined at two different time points. Second, in between the twomeasurements, a hypertonic glucose-amino acid solution was infused. Thecombination of these two procedures seemed to enhance the detection ofmalignant disease with polyamine measurements.

Increases in RBC putrescine during TPN are thus related to eitherincreased substrate levels with constant tumor ODC activity or increasedtumor ODC activity and subsequent proliferation. In addition it appearsthat increases in RBC polyamines during TPN in cancer patients arerelated to tumor proliferation.

An important concept of polyamine metabolism is that tumors may have agreater requirement for polyamine synthesis, whereas normal tissues mayhave a lesser requirement for polyamines because of their controlledgrowth behavior. By providing nutrient substrates, TPN may be increasingpolyamine production in tumor cells that then excrete these productsinto the extracellular space and are absorbed by RBC.

In particular, the TPN solution utilized in the previous example wasTravasol 10%, whose amino acid composition is displayed in Table 1.Travasol 10% is used in compounding a feeding solution for generaladministration in patients with benign or malignant diseases. However,the present inventors contemplate that any of the commercially availableparenteral amino acid formulations will function in the practice of thepresent invention. In fact, amino acid formulations may be designedwhich include a wide range of amino acid concentrations andcombinations. For example, parenteral solutions with up to three timesthe amino acid concentration of commercial TPN solutions would not beconsidered toxic and would therefore be functional in the presentinvention. It appears that the only requirement for practicing thepresent invention is that such solutions contain one or more of theessential amino acids and either ornithine or arginine, or both, withornithine being preferred.

EXAMPLE II: FEEDING SOLUTIONS CONTAINING ORNITHINE

Ornithine has been combined with essential amino acids and utilized in arat tumor model to demonstrate its potential utility in man. Rats havingbeen implanted with a methylcholanthrene (MCA)--induced tumors receivedcontinuous infusion of one-fold diluted Nephramine 5.4% in combinationwith either arginine or ornithine. Polyamine levels were determined bothbefore and after feeder solutions infusion in control no arginine orornithine experimental (tumor-bearing rats given solutions withornithine or arginine) and sham or chow-fed rats.

Male Fisher 344 rats were purchased from Timco Harlan-Sprague-Dawley(Houston, Tex.). All rats were allowed a 7-day acclimation period withchow (Purina 5001) and water ad libitum. A methylcholanthrene-inducedfibrosarcoma (0.17 g) was implanted as a brei into the right flank underanesthesia and the animals were fed a chow diet for 21 days. When thetumors were at least 1.0 cm in width, two-dimensional measurements withcalipers were instituted and the equation, length (cm)×(width [cm])²×1/2=grams of tumor tissue, was used to estimate tumor weight. Whentumor weight reached 14±3 g (21 days of chow diet), the animals wererandomized into three groups by tumor weight and under anesthesia aSilastic central venous catheter was placed in the superior vena cavathrough an internal jugular vein cutdown. The animals were then allowedto recover overnight and continuous infusions were started the nextmorning. Essential amino acids+arginine rats (E+A rats) received acontinuous infusion of 500 ml 60% glucose+500 ml 5.4% Nephramine with0.58 g/100 ml or arginine added for 6 days. Essential aminoacids+ornithine rats (E+0 rats) received 500 ml 60% glucose+500 ml 5.4%Nephramine with 0.44 g/100 ml of ornithine added. The ornithine andarginine were added at equimolar concentrations. Nephramine is aparenteral amino acid solution consisting of eight essential amino acidsand histidine. After 6 days of continuous infusion, the animals weresacrificed by aortic bleeding. Liver and tumor were excised and weighedand kept frozen at -70° C. until assays were done. Only viable,nonnecrotic tumor tissue was saved and assayed.

Preparation of tumor tissue for polyamine assay was done in thefollowing manner. Tumor tissue weighing 0.5 to 1.5 g was cut into smallpieces and put into a disposable tube (16×100 mm). Ice-cold 4%sulfosalicylic acid was then added at 2 ml/g wet tissue. This mixturewas homogenized in an ice bath for 40 seconds using the BrinkmanPolytron homogenizer (P10ST generator set at 8.5). The suspension wascentrifuged at 100,000 g for 30 minutes and the resulting clearsupernatant was analyzed for polyamines on a Durrum D-500 amino acidanalyzer.

Statistical analyses were done with Student's t test (one tail). RBCdeterminations were performed as described above; blood was obtained byaortic puncture.

The results are demonstrated in Table IV. As Table IV indicates, levelsof putrescine within the tumors were increased approximately three foldwhereas levels of all three polyamines were significantly increased inthe rat's RBC's. In both cases, ornithine functioned better thanarginine, but both appeared to promote an increase in tumor-relatedlevels. Comparison of these valves from tumor-bearing rats to non-tumorbearing would demonstrate an even more substantial difference inrelative polyamine levels.

                  TABLE IV                                                        ______________________________________                                        Nutritional                                                                              Tumor Weight.sup.1                                                 Regimen.sup.1                                                                          n     Initial     Final   % Change.sup.2                             ______________________________________                                        Chow     5     13.3 ± 2.6                                                                             32.9 ± 5.3                                                                         134 ± 32                                E        4     14.0 ± 2.8                                                                             35.7 ± 6.9                                                                         135 ± 42                                E + A    6     10.5 ± 2.2                                                                             25.7 ± 3.4                                                                         153 ± 68                                E + O    7     11.1 ± 2.2                                                                             24.4 ± 4.9                                                                         123 ± 34                                ______________________________________                                        Nutritional   Polyamine Levels                                                Tissue                                                                              Regimen   n     Putrescine                                                                            Spermidine                                                                             Spermine                               ______________________________________                                        Eryth-                                                                              Chow      5     0.91 ± 0.21                                                                         71 ± 57                                                                             6.1 ± 4.1                          rocyte                                                                              E         4     1.42 ± 0.32                                                                        NA       NA                                           E + A     6     1.60 ± 0.52                                                                         69 ± 45                                                                             6.5 ± 2.6                                E + O     7     1.85 ± 0.36                                                                        125 ± 9                                                                             10.9 ± 3.3                          Tumor Chow      5     46.9 ± 12.2                                                                        797 ± 80                                                                            392 ± 21                                  E         4     22.9 ± 4.4                                                                         901 ± 39                                                                            480 ± 35                                  E + A     6     50.5 ± 13.3                                                                        871 ± 69                                                                            409 ± 36                                  E + O     7     73.6 ± 17.1                                                                        919 ± 90                                                                            415 ± 30                            ______________________________________                                         .sup.1 Fibrosarcome, growing s.c.                                             .sup.2 % increase in tumor weight.                                       

Additionally, the previous example demonstrates that 450 mg of ornithinefor every 100 ml of the amino acid formulation functions satisfactorilyand continuous infusion of such solutions which posed no toxicityproblems. It is contemplated that amino acid solutions ranging from 50mg up to 2 g per 100 ml of solutions (with between 50 and 750 mg/100 mlbeing a preferred range for arginine and ornithine) should functionsatisfactorily without loss of appreciable activity or increasedtoxicity. However, for the detection of microscopic tumors and as anantidote to ODC-directed antitumor therapy, higher concentrations ofornithine are suggested.

Another experiment which utilized formulations containing amino acidprecursors of RBC polyamines, compared the response of tumor-bearingrats (TB) to non tumor-bearing rats (NTB). The study was performedbasically in the manner described above with the following indicateddifferences. Fibrosarcoma-bearing (TB) and non-tumor being F344 rats(NTB) were infused with either a typical feeding solution (30%glucose+5% essential amino acids), with essential amino acids (19 g/L,30% glucose) plus arginine at 19.5% g/l (E+A). Chow rats (C) were fedchow with no i.v. infusion.

                  TABLE V                                                         ______________________________________                                                      NTB                        TB                                   Treatment                                                                             N     PUT     N   WT.  PUT   N   WT   PUT                             ______________________________________                                        C       13    .39 ±                                                                              5   13 ±                                                                             .7 ±                                                                            6   37 ±                                                                            1.2 ± 0.2                                  .10         3    10        10                                   E        9    .41 ±                                                                              6   15 ±                                                                             .9 ±                                                                            4   36 ±                                                                            1.4 ± 0.3                                  .07         2    .3        7                                    E + A    5    .62 ±                                                                              8   11 ±                                                                            1.9 ±                                                                            2   51 ±                                            .22         3    .3        22                                   5.0 ± 2.7                                                                  ______________________________________                                    

Where N is the number of rats included in the study, PUT is RBC levels(nm/g/ml, means±SD) of putrescine determined by HPLC.

As demonstrated in Table IV, the increase in RBC putrescine levels inNTB rats following administration of feeding solutions which included aspecific putrescine precursor (arginine) was significantly greater(p<0.05) than those receiving chow or essential amino acids only.However, the RBC putrescine level of TB rats receiving E+A wassignificantly greater than any other group in the study. It should alsobe noted that increased putrescine levels were found to be proportionalto tumor weight.

c. REDUCTION OF DFMO INDUCED THROMBOCYTOPENIA

Animal and clinical studies have shown that the major host toxicityassociated with the continuous infusion of DFMO is thrombocytopenia (Otaet al. (1986), Int. J, Cancer, 38:245; see also FIG. 3 herein). In theabove described rat model, a continuous infusion of DFMO was giventhrough a central venous catheter. Non-tumor-bearing and tumor-bearingrats received continuous i.v. DFMO for 12 days. The DFMO doses typicallyemployed were 500 mg, 1000 mg, and 2000 mg per kg body wt per day.Although there was histological evidence of small intestinal mucosalatrophy at these doses, no significant clinical toxicity was seen duringthe 12 day study. However, dose related thyrombocytopenia was observed,as shown in FIG. 3. White cell count and hematocrit changes were notsignificantly altered by DFMO administration.

Clinical studies with patients undergoing DFMO therapy have shown thatthe continuous infusion of DFMO for 28 days also produced significantclinical thrombocytopenia. Eleven cycles of 28 day DFMO infusion havebeen administered to 7 patients at doses of either 6 or 8 g DFMO/m²/day. As shown in FIG. 4, the platelet suppression was significant atboth doses. There was significantly greater platelet suppression at the8 g dose as compared with the 6 g dose. Significant suppression of thehematocrit was also noted with a trend toward dose related suppression.White cell count was not affected by either dose. There was no incidenceof nausea, vomiting or diarrhea. There were two patients who experienceddecreased hearing acuity at 8 h DFMO/m² /day. All toxicities resolvedspontaneously within two weeks of discontinuing the DFMO infusion.

The following study was designed to determine if the simultaneousadministration of ornithine with DFMO would reduce thrombocytopenia.Central venous catheters were inserted into Fisher 344 male rats. Theanimals received approximately 1500 mg DFMO per kg body wt per day as acontinuous i.v. infusion. Ornithine HCl (Ajinomoto, Ltd., New York,N.Y.) was added to the saline solutions at a final concentration of 3.3mmol/100 ml. This concentration is equimolar to the arginineconcentration in 10% Travasol. After 12 days of infusion the rats weresacrificed and platelet counts were determined in peripheral blood.

Table IV shows the results of this experiment. There were 3 treatmentgroups consisting of saline, DFMO and DFMO with ornithine. As shown inTable VI, the simultaneous administration of ornithine with DFMO blockedplatelet suppression. DFMO alone resulted in a significant decrease inplatelet count as compared with the saline treatment.

                  TABLE VI                                                        ______________________________________                                        Influence of Ornithine                                                        Co-administration on DFMO-Induced                                             Thrombocytopenia in the Rat                                                   Treatment     n     Platelets (× 10.sup.3 /cu. mm)                      ______________________________________                                        Saline        3     780 ± 253                                              DFMO.sup.b    5     409 ± 127                                              DFMO + Orn.sup.c                                                                            6     .sup. 958 ± 195.sup.d                                  ______________________________________                                         .sup.a Fischer 344 male rats received a continuous infusion of saline,        DFMO or DFMO + ornithine for 12 days. All rats received chow and water ad     libitum.                                                                      .sup.b DFMO dose = 1444 ± 73 mg/kg/d.                                      .sup.c DFMO dose = 1429 ± 85 mg/kg/d; Ornithine dose = 436 mg/100 ml o     3.3 mmoles/100 ml infusion.                                                   .sup.d Mean differs significantly compared with DFMO treated rats.       

These results demonstrated that parenteral administration of polyamineprecursors can significantly reduce DFMO induced platelet toxicity. Theresults further indicate that, at least in the animal system studied, aconcentration of approximately 33 mM is adequate to prevent or reducethe occurrence of thrombocytopenia. However, it is contemplated thatvirtually any non-toxic amount of polyamine precursor can be employed.Moreover, the results indicate that polyamine precursors may beadministered alone and do not require the presence of additional aminoacids.

d. INHIBITION OF TUMOR GROWTH

An important consideration in developing a tumor-inhibiting formulationis the interaction between tumor versus host polyamine utilization andspecific amino acid solutions. It has been determined thatadministration of standard TPN solutions to patients with occultmalignancies produce an elevation in polyamine levels compared withnon-cancer patients. Consequentially, this phenomenon has been used inthe formulation of specific amino acid solutions for detecting thepresence of tumors in patients.

Generally, reduced levels of tumor polyamines have been correlated to adecrease in tumor growth rate. However, infusion of amino acid solutionsrich in ornithine will increase polyamine levels despite the decrease intumor growth rate elicited. It is hypothesized that this anomoly may beexplained by ornithine's status as a non-essential amino acid. proteins,thus possibly explaining the lack of any increase in tumor growthexhibited in animals receiving formulations rich in ornithine.Additionally, ornithine is only passively taken up by the cells,compared to the active uptake of essential amino acids such as arginine.Also, the high polyamine levels might be explained by ornithine's statusas a polyamine precursor. The reasons for the relatively large standarderrors in the ornithine group tumor studies are currently beinginvestigated to determine the actual role of ornithine in tumor growth.

In recent years, several studies in animal-tumor models have shown thatnutritional therapy can result in tumor growth and, in some cases,accelerate growth. For some tumors, tumor growth can also occur duringhost starvation, emphasizing that tumors will obtain either exogenous orendogenous nutrients for their energy needs and biosynthetic pathways.(Sauer, L. A., Nagel, W. D., Dauchy R. T., et al. (1986), Cancer Res.,46:3469.

One strategy in approaching the problem of host-tumor competition fornutrients is to formulate an amino acid regimen that does not includesubstrates that enhance tumor growth. The present invention addressesthis strategy by defining specific combinations and concentrations ofamino acids that decrease the presence of amino acids that are polyaminebiosynthetic precursors hypothesized as being responsible forstimulating tumor growth. A formula which includes a non-toxic,pharmacologically acceptable amount of the amino acids together withornithine and citrulline and less than 0.45% by weight finalconcentration of arginine is one formula which has been found to inhibittumor growth.

It is hypothesized that arginine is the specific amino acid responsiblefor stimulating tumor growth. Arginine is commonly found in generalparenteral amino acid formulas and by weight (mg/100 ml) makes up 10% ofthe amino acid composition before formulation. After formulation of sucha general parenteral amino acid formula, arginine comprises about 1% byweight final concentration (1.0 g./100 ml). Applicants' solutionsdecrease or eliminate this standard arginine content. Also, ornithineand citrulline are not present in any commercially available parenteralamino acid formula. These amino acids are necessary to Applicant'sformulas. Thus, standard solutions could not conveniently be used intheir commercially available concentrations to practice the Applicantsmethods or formulations.

The subject invention includes several formulations comprised of variousmixtures of essential and non-essential amino acids obtained incrystalline form. Arginine may or may not be included in the particularformula. A sufficient quantity of citrulline or ornithine is then addedso as to constitute greater than about 0.5% by weight concentration ofthe solution upon the addition of a suitable liquid medium. Onepreferred liquid medium is a glucose solution. This glucose solution mayrange from 5% to 70% glucose in the present formulations. Patientnutritional support formulations are commonly constituted in suchglucose solutions. Desirable relative internal proportions of theincluded amino acids are defined by the following Table VII for every100 milliliters of the formulation.

                  TABLE VII                                                       ______________________________________                                                  Amino Acids                                                                             Mg./100 ml. formulation                                   ______________________________________                                        Essential   leucine     250 to 1400 mg                                        Amino Acids isoleucine  200 to 1400 mg                                                    valine      200 to 1250 mg.                                                   phenylalanine                                                                             100 to 900 mg                                                     methionine   50 to 850 mg                                                     lysine      150 to 750 mg                                                     histidine    85 to 500 mg                                                     threonine   100 to 550 mg                                                     tryptophan   50 to 200 mg                                         Non-essential                                                                             alanine     200 to 3500 mg                                        Amino Acids glycine     250 to 2000 mg                                                    proline     100 to 1500 mg                                                    serine       5 to 650 mg                                                      tyrosine     30 to 60 mg                                          ______________________________________                                    

It will be understood that in addition to the amino acids, theformulation may include preservative agents.

The preparations may be advantageously prepared in the form of sterileaqueous solutions adapted for intravenous administration. In accordancewith known practice for such solutions, the malignant disease amino acidsolutions will be sterile, pyrogen free, and at a suitable pH forparenteral administration.

Specific formulations for practicing the present invention are set outin the following examples.

EXAMPLE III

A sterile, non-pyrogenic, stable solution suitable for parenteraladministration to patients with malignant disease is prepared from purecrystalline amino acids, which are dissolved in a glucose solution (5%to 70%) in the following concentrations:

    ______________________________________                                        Amino Acids      Mg./100 ml. formulation                                      ______________________________________                                        leucine          250 to 1400 mg                                               isoleucine       200 to 1400 mg                                               valine           200 to 1250 mg.                                              phenylalanine    100 to 900 mg                                                methionine        50 to 850 mg                                                lysine           150 to 750 mg                                                histidine         85 to 500 mg                                                threonine        100 to 550 mg                                                tryptophan        50 to 200 mg                                                tyrosine          30 to 60 mg                                                 ______________________________________                                    

This formulation is arginine-free.

To the foregoing formulation is added a sufficient quantity ofcitrulline or ornithine so as to constitute about 1% by weight of theformulation. At least one of the following non-essential amino acids isthen added in the following concentration:

    ______________________________________                                        Amino Acids      Mg./100 ml. formulation                                      ______________________________________                                        alanine          200 to 3500 mg                                               glycine          250 to 2000 mg                                               proline          100 to 1500 mg                                               serine           °5 to 650 mg                                          ______________________________________                                    

The solution is then filtered into appropriate containers forintravenous fluids and steam sterilized at 250° F. for 10 minutes. Toprepare for administration, the volume is then brought to the desiredfeeding solution volume with an additional volume of glucose solutionand kept cool. The solution is then administered to the patientintravenously (i.v.).

EXAMPLE IV

If a formulation of amino acids for patients with malignant disease isdesired which contains ornithine, but is citrulline free, the formula asoutlined in Example III can be utilized. Ornithine will be added toconstitute greater than about a 0.5% by weight concentration of theformulation, and no citrulline will be added. The same proportions ofthe essential and non-essential amino acids (leucine, isoleucine,valine, phenylalanine, lysine, valine, isoleucine, threonine,tryptophan, histidine, tyrosine, alanine, glycine, proline and serine)will be present; and the solution prepared in the same manner. Thisformulation is arginine-free.

EXAMPLE V

Following the procedure of Example IV, an alternate amino acidformulation with arginine for patients with malignant disease isprepared from the following pure crystalline amino acids and in thefollowing concentrations:

    ______________________________________                                                  Amino Acids                                                                             Mg./100 ml. formulation                                   ______________________________________                                        Essential   leucine     250 to 1400 mg                                        Amino Acids isoleucine  200 to 1400 mg                                                    valine      200 to 1250 mg.                                                   phenylalanine                                                                             100 to 900 mg                                                     methionine   50 to 850 mg                                                     lysine      150 to 750 mg                                                     histidine    85 to 500 mg                                                     threonine   100 to 550 mg                                                     tryptophan   50 to 200 mg                                         Non-essential                                                                             alanine     200 to 3500 mg                                        Amino Acids glycine     250 to 2000 mg                                                    proline     100 to 1500 mg                                                    serine       5 to 650 mg                                                      tyrosine     30 to 60 mg                                          ______________________________________                                    

This mixture of essential and non-essential amino acids is thendissolved in distilled water. To the foregoing formulation is added asufficient quantity of arginine so as to constitute less than about0.10% by weight of the final formulation. Ornithine or citrulline isthen added so as to constitute at least about 0.50% by weight of thefinal formulation. The volume is then brought to the desired volume witha glucose solution (5% to 70%). The solution is then filtered intoappropriate containers for intravenous fluids and steam sterilized at250° F. for 10 minutes.

To prepare the feeding solution, the stock formula above is then addedto an appropriate volume of glucose solution (5% to 70%) so as toconstitute a final arginine concentration of less than about 0.10% byweight.

EXAMPLE VI

If a formulation of amino acids for patients with malignant disease isdesired which contains alanine as the non-essential amino acid ofchoice, the formula as outlined in Example III can be utilized. The sameproportions of the essential amino acids (leucine, isoleucine, valine,phenylalanine, methionine, lysine, histidine, threonine, tryptophan andtyrosine) will be present; and the solution prepared in the same manner.Alanine will then be included at about a 1% by weight finalconcentration of the formula upon the addition of a glucose solution tothe prepared stock.

EXAMPLE VII

A sterile, non-pyrogenic, stable solution suitable for parenteraladministration to patients with malignant disease is prepared from purecrystalline amino acids, which are dissolved in distilled water in thefollowing concentrations:

    ______________________________________                                                  Amino Acids                                                                             Mg./100 ml. formulation                                   ______________________________________                                        Essential   leucine     250 to 1400 mg                                        Amino Acids isoleucine  200 to 1400 mg                                                    valine      200 to 1250 mg.                                                   phenylalanine                                                                             100 to 900 mg                                                     methionine   50 to 850 mg                                                     lysine      150 to 750 mg                                                     histidine    85 to 500 mg                                                     threonine   100 to 550 mg                                                     tryptophan   50 to 200 mg                                         Non-essential                                                                             alanine     200 to 3500 mg                                        Amino Acids glycine     250 to 2000 mg                                                    proline     100 to -500 mg                                                    serine       5 to 650 mg                                                      tyrosine     30 to 60 mg                                          ______________________________________                                    

To the foregoing formulation is added a sufficient quantity of ornithineso as to constitute about greater than 0.5% by weight of the finalformulation. Also, citrulline is to be added so as to constitute about0.01 to 2% by weight of the final formulation. Arginine is to beincluded so as to constitute a less than about 0.10% by weightconcentration of the final formulation. The solution is then filteredand filled into appropriate containers for intravenous fluids and steamsterilized at 250° F. for 10 minutes. To prepare the actual feedingsolution from the above stock, the volume is then brought to the desiredvolume with a glucose solution so as to constitute at least a 0.50% byweight ornithine and less than about 0.10% arginine.

To test the above amino acid formula theory, Applicants conducted apreliminary study employing two different regimens, one a standard TPNsolution, the other a control group. Each regimen was administeredparenterally to adult rats for a period of 12 days. These rats had priorto treatment were implanted with transplantable colon tumor in the Rflank. When the implant had grown to approximately 3 grams, therespective regimen was administered through a central venous catheter.As shown in FIG. 5, tumor growth in weight for the TPN group wassignificantly greater than the CHOW final tumor weight (P<0.05).a=p<0.05 compared with CHOW final tumor weight.

The following studies, Examples VIII IX, and X were designed todetermine if the polyamine precursors in TPN solutions were responsiblefor stimulating tumor growth. In Example VIII, Fisher 344 male ratsreceived an implant of Ward Colon carcinoma (WCC) in the R flank. Whenthe implants had grown to approximately 3 grams, the rats wererandomized to four groups, and a central venous catheter inserted. Fourdifferent regimens, including a standard TPN regimen, an EAA (essentialamino acid) regimen, a EAA +NEAA (essential amino acids plusnonessential amino acids arginine free) and a Control group (i.v. salineand chow ad libitum), were infused for 12 days through the catheter. Thecontent of each regimen is presented at Table VIII. As shown in FIG. 8,the greatest rate of tumor growth was exhibited in the TPN group. Thisstudy suggested arginine may be important in stimulating tumor growth.

Example IX was then devised to confirm the results in the previous studythat suggested the presence of arginine in TPN solutions contributed totumor growth. Fisher 344 male rats first received an implant of WardColon carcinoma. When the implant grew to approximately 4 grams, theanimals were randomized into four groups and a central venous catheterinserted. Arginine and ornithine were then added back to separate TPNformulas. A third regimen wherein alanine was added back to theformulation was also used. A control group received saline intravenously(i.v.) and chow ad libitum. Each formulation was infused to a separategroup of the above described rats. The contents of each of theseregimens is outlined in Table IX. The respective solutions wereparenterally administered for 6 days, after which time tumor growth wasassessed. The results, as shown at FIG. 9, indicate that the addition ofarginine increased WCC growth compared with control. This is in contrastto the results obtained when either alanine or ornithine was added tothe TPN solution, where there was observed no significant difference intumor growth compared with control.

Example X was then conducted to determine the concentration of argininewhich increased tumor growth rate. Fisher 344 male rates were preparedas above and then randomized to four groups. Three arginine regimenswith decreasing concentrations of arginine were formulated as outlinedin Table X. A control group received saline and chow ad libitum. After12 days of parenteral administration of the various regimens, tumorgrowth and urine amonia levels were assessed. The results, as shown inFIGS. 10 and 11, indicate that concentrations of arginine as low as0.10% by weight were able to stimulate tumor growth rate over controls.Urine ammonia levels were found to be inversely proportional to arginineregimen concentration, with the highest urine amonia levels present inthe 0.10% arginine group. No detectable urine amonia was present in theurine of control group animals. These results suggest that argininestimulates tumor growth rate and that a non-stimulatory concentration ofarginine is most preferably less than 0.10% by weight finalconcentration.

These results indicate that cancer patients requiring TPN would benefitfrom a modified amino acid formula that deletes or reduces arginine.Additionally, a method of inhibiting tumor growth using formulationsthat contain less than about 0.10% by weight arginine content is hereindevised. Ornithine and/or citrulline should be added to these modifiedsolutions in a concentration sufficient to maintain substrates for theurea cycle. Ornithine in at least a 0.5% by weight final concentrationof the feeding formulation is adequate for urea cycle substratepurposes. The results obtained in the following experiments demonstratethat a specific amino acid formula with decreased arginine concentration(less than about 0.10% by weight) can effectively reduce tumor growthrate during nutritional therapy in a patient with malignant disease.

EXAMPLE VIII

This study was performed to demonstrate that the presence of particularprecursors for polyamine biosynthesis in TPN solutions is responsiblefor stimulating tumor growth.

Male Fisher 344 rats were purchased from Timco Harlan-Spraque-Dawley(Houston, Tex.). All rats were allowed a 7-day acclimation period withchow (Purina 5001) and water ad libitum. A Ward colon carcinoma (WCC)was implanted subcutaneously in the right flank of each animal underanesthesia. When these tumor implants had grown to approximately 3grams, the rats were randomized to four groups and a central venouscatheter inserted in the right flank. Each group of rats received one ofthe following 4 formulations: TPN; EAA (Essential Amino Acid Solution)with arginine; EAA+NEAA (Essential Amino Acid and Nonessential AminoAcid solution), without arginine; or Control (Saline). The control groupof five rats received saline by infusion and rat chow. All solutionswere administered for 12 days. In particular, the amino acid compositionof the parenteral regimens used in this example is displayed in TableVIII. The TPN solution utilized in formulating a solution for one of thegroups was Travesol 10% whose amino acid composition is displayed inTable 1. Travesol 10% stock is used in compounding a feeding solutionfor general administration in patients with benign or malignantdiseases.

Tumor weight was determined in each group before and after the 12-daytreatment period. Results from this study are shown in FIG. 8. TheControl group exhibited a tumor weight of 11±r while the TPN treatedgroup exhibited a tumor weight of 39±2 g. There was no significantincrease in tumor weight in the group receiving the EAA regimen (11+2).The EAA+NEAA arginine-free treated rats exhibited a slight increase intumor weight, but much less than the TPN treated group (17 +4). The dataindicates that the TPN-stimulated tumor growth may be controlled byrestricting the arginine content.

ANIMAL POPULATION

Male Fisher 344 rats were purchased from Timco Harlan-Sprague-Dawley(Houston, Tex.). All rats were allowed a 7-day acclimation period withchow (Purina 5001) and water ad libitum. Each rat was then inoculatedsubcutaneously in the right flank with a transplantable Ward Coloncarcinoma (WCC). When the implants grew to approximately 3 grams inweight, the rats were randomized to four groups.

NUTRITIONAL REGIMEN

The content of the solutions used in this study appear at Table VIII.TPN solutions consisted of 500 ml D50W+500 ml 10% Travesol. EAA regimenconsisted of 500 ml D50W+500 ml essential amino acid (EAA) solution. EAA+NEAA consisted of 500 ml D50W+500 ml essential and non-essential aminoacids (NEAA) deleting arginine. Control received intravenous saline andchow ad libitum. All solutions were administered through a centralvenous catheter by continuous i.v. infusion for 12 days. Daily intake ofcontrol rats was 52±9 cal and 584±104 mg nitrogen. Daily intake of TPNwas 54±2 cal and 550±5 mg nitrogen. EAA daily intake was 51+4 cal and270±10 mg nitrogen. EAA+NEAA regimen daily intake was 51±5 cal and574±15 mg nitrogen. TPN and EAA+NEAA regimens were isonitrogenous andisocaloric with the only difference being the content of arginine.

STUDY DESIGN

Each of 18 Fisher adult male rats were given a subcutaneous implantation(in the right flank) of a fixed volume of tumorous cells. When theimplant of tumorous Ward colon carcinoma cells reached about a 3 gramweight, the rats were randomized to four groups and a central venouscatheter inserted. Each group was to receive either TPN, EAA formula,EAA+NEAA (arginine free) or Saline (control group) for a period of 12days. Calculations of each tumor weight in grams in each of the animalswas made after the 12 day regimen. In this manner, each rat served asits own control in determining the effect of the particular regimen ontumor growth. Analysis of data was done by a student's t-test, comparingtumor growth before and after the designated regimen between treatmentgroups.

TUMOR GROWTH DETERMINATIONS

The TPN-induced tumor growth was determined from the calculated tumorweight in grams after the 12-day treatment. The results are shown inFIG. 8. Tumor growth was assessed by two-dimensional measurements withcalipers, (length and width) and the equation, length (cm) ×(width [cm]²×1/2=grams of tumor tissue, was used to estimate tumor weight.

The results are demonstrated in FIG. 7. As FIG. 8 indicates, TPNresulted in tumor growth stimulation compared with Control. EAA did notstimulate tumor growth compared with Control. There was a slightincrease in tumor growth with EAA+NEAA compared with Control, but tumorsize was markedly lower than TPN tumors. The EAA+NEAA regimens did notcontain arginine, thus, it is deduced that the presence of arginine inTPN solutions is important in stimulating tumor growth. TPN solutionscontain final arginine concentrations of about 1% by weight in thefeeding formulations.

                  TABLE VIII                                                      ______________________________________                                        Amino Acid Composition of                                                     Parenteral Regimens of Rats                                                                   Concentrations of each amino                                  Amino           acid(mg) per 100 ml                                           Acid         % N    EAA      EAA + NEAA                                                                              TPN                                    ______________________________________                                        LEUCINE      10.67  825      825       730                                    METHIONINE   9.39   825      825       400                                    PHENYLALANINE                                                                              8.48   825      825       560                                    LYSINE       19.16  600      600       580                                    VALINE       11.96  600      600       580                                    ISOLEUCINE   10.68  525      525       600                                    THREONINE    11.76  375      375       420                                    TRYPTOPHAN   13.72  188      188       180                                    HISTIDINE    27.08  412      412       480                                    ALANINE      15.72  --       3210      2070                                   ARGININE     32.16  --       --        1510                                   GLYCINE      18.66  --       1599      1030                                   PROLINE      12.17  --       1059      680                                    SERINE       13.33  --       780       500                                    TYROSINE     7.73   --        51        40                                    ______________________________________                                         The EAA regimen was formulated with 500 ml of Aminess ®, 500 ml D50W,     electrolytes, and multivitamins. EAA + NEAA regimen was formulated with       500 ml of 10% EAA + NEAA, 500 ml D50W, electrolytes, and multivitamins.       TPN regimen consisted of 500 ml 10% Travasol, 500 ml D50W, electrolytes,      and multivitamins. These were administered by continuous infusion through     a central venous catheter, the flow rate being controlled with Holter 903     pump. The amino acid concentration (mg/100 ml) in this table is the           concentration of the 500 ml amino acid solution before formulation.           Formulation was accomplished by the addition of a glucose solution to the     above described stock.                                                   

EXAMPLE IX

This study was performed to determine whether the addition of arginineand ornithine to a total parenteral nutritional (TPN) regimen wouldstimulate tumor growth. TPN regimens containing alanine, arginine orornithine were administered i.v. A control group received saline andchow ad libitum. The various regimens were administered parenterally tomale Fisher 344 rats with Ward Colon Carcinoma implants that had grownto approximately 4 grams. Each group consisted of 5 rats. Tumor size wasdetermined before and after the designated regimen for each animal.Percent increase in tumor weights in the ALANINE regimen group and theORNITHINE regimen group did not differ from the CONTROL group. Anincrease in percent tumor growth compared with Control was observed inthe ARGININE group, yet no significant difference was observed in theALANINE group. The data indicates that cancer patients requiring TPNwould benefit from a modified amino acid formula that deletes or reducesarginine content.

ANIMAL POPULATION

Male Fisher 344 rats were purchased from Timco Harlan-Sprague-Dawley(Houston, Tex.). All rats were allowed a 7-day acclimation period withchow (Purina 5001) and water ad libitum. A Ward Colon Carcinoma (WCC)was implanted subcutaneously in the right flank of each rat underanesthesia. When the implant grew to approximately 3 grams, the ratswere randomized to four groups. A central venous catheter was theninserted into each rat.

NUTRITIONAL REGIMEN

Four nutritional regimens were formulated for this study. Each group ofrats were given one of these regimens.

The amino acid composition of each regimen is displayed at Table IX. TheALANINE regimen consisted of 500 ml D50W+250 ml 5.5% essential aminoacid (EAA, Nephramine)+294 mg/100 ml alanine. ARGININE regimen consistedof 500 ml D50W+250 ml 5.5% EAA+575 mg/100 ml arginine. ORNITHINE regimenconsisted of 500 ml D50W+250 ml 5.5% EAA+436 mg/100 ml ornithine. TheCONTROL regimen consisted of saline infusion with chow supplied adlibitum. Table IX defines the composition of each of these formulations.The solutions were infused for 6 days and tumor growth was assessed.

STUDY DESIGN

Each of 20 Fisher adult male rats were tiven a subcutaneous implantationin the right flank of a fixed volume of tumorous cells, Wards coloncarcinoma. When the implant grew to approximately 3 grams, the rats wererandomized into 4 different treatment groups. A central venous catheterwas then inserted. Each group was to receive a regimen of ALANINE,ORNITHINE, ARGININE, or SALINE (CONTROL) and chow ad libitum for 6 days.Tumor growth in each animal was assessed for percent increase in tumorweight compared to that animals pre-regimen tumor weight. In thismanner, each rat served as his or her own control in determining theeffect of the particular regimen on tumor growth. The various treatmentregimens were also compared as against the CONTROL group for percenttumor growth.

PERCENT TUMOR GROWTH DETERMINATIONS

Tumor growth was assessed by a measure of percent increase in tumorweight before and after the designated regimen. The results aredemonstrated in FIG. 8. As demonstrated in FIG. 8, the ALANINE formuladid not stimulate WCC tumor growth compared with CONTROL. Increased WCCtumor growth compared with CONTROL was observed when ARGININE wassubstituted for alanine at an equimolar amount. There was no significantdifference in tumor growth compared with CONTROL when TPN formulacontained ORNITHINE.

                  TABLE IX                                                        ______________________________________                                        Amino acid composition of                                                     parenteral regimens of rats                                                              Concentration of each amino                                        Amino      acid(mg) per 100 ml                                                Acid       ALANINE   ARGININE    ORNITHINE                                    ______________________________________                                        LEUCINE    293       293         293                                          METHIONINE 293       293         293                                          PHENYL-    293       293         293                                          ALANINE                                                                       LYSINE     213       213         213                                          VALINE     213       213         213                                          ISOLEUCINE 187       187         187                                          THREONINE  110       110         110                                          TRYPTOPHAN  55        55          55                                          HISTIDINE  143       143         143                                          ALANINE    294        0           0                                           ARGININE    0        575          0                                           ORNITHINE   0         0          436                                          ______________________________________                                         The final regimens were formulated with 250 ml of the above amino acid        solution, 500 ml D50W, TPNelectrolytes (Abbott Laboratories), and             multivitamins. These were administered by continuous infusion through a       central venous catheter, the flow rate being controlled with Holter 903       pump. Concentration of amino acid represents final concentration in total     volume of 750 ml. Alanine, arginine, and ornithine were added in equimola     amounts.                                                                 

EXAMPLE X

This study was performed to determine the concentrations of argininewhich stimulate tumor growth. Modified TPN solutions containing 0.10%,0.25% or 0.65% Arginine by weight final feeding formulationconcentrations were administered i.v. A control group received i.v.0.90% NaCl (saline) and chow ad libitum. The various regimens wereadministered parenterally to male Fisher 344 rats with Ward ColonCarcinoma implants that had grown to approximately 4 grams. Each of theArginine groups consisted of 4 rats while the control group consisted of3 rats. Tumor size was determined before and after the designatedregimen for each animal. Urine ammonia levels were also determinedbefore and after the designated regimen for each animal. Thepost-treatment tumor weights (g) in the 0.10%, 0.25% and 0.65% Argininegroups did differ significantly from the Control group. However, nosignificant difference was observed in post-treatment tumor weightsbetween the various Arginine groups. The data indicates that patientsrequiring TPN would benefit from a modified amino acid formula thatdeletes or reduces arginine content to less than 0.10% by weight finalconcentration.

ANIMAL POPULATION

Male Fisher 344 rats were purchased from Timco Harlan-Sprague-Dawley(Houston, Tex.). All rats were allowed a 7-day acclimation period withchow (Purina 500) and water ad libitum. A Ward Colon Carcinoma (WCC) wasimplanted subcutaneously in the right flank of 15 rats under anesthesia.When the implant grew to approximately 4 grams, the rats were randomizedto 3 groups of 4 rats each and 1 group of 3 rats. A central venouscatheter was then inserted into each rat.

NUTRITIONAL REGIMEN

Four nutritional regimens were formulated for this study. Each group ofrats was given one of these regimens.

The amino acid composition of each regimen is displayed at Table X. The0.65% arginine regimen consisted of 500 ml D50W+500 ml Aminess®⁺ 0.65g./100 ml. arginine. The 0.25% arginine regimen consisted of 500 mlD50W+500 ml Aminess®+0.25 g./100 ml arginine. The 0.10% arginine regimenconsisted of 500 ml D 50W+500 ml Aminess®+0.10 g./100 ml arginine. TheCONTROL regimen consisted of saline infusion with chow ad libitum. TableX also defines the mixture and proportion of other amino acids containedin each of the three arginine regimens. The solutions were infused for12 days. Tumor growth and urine ammonia levels were then assessed andcompared to pre-treatment levels.

STUDY DESIGN

Each of 15 Fisher adult male rats were given a subcutaneous implantationin the right flank of a fixed volume of tumorous cells, Wards ColonCarcinoma. When the implant grew to approximately 4 grams, the rats wererandomized into 4 treatment groups. A..central venous catheter was theninserted. Each group was to receive a regimen of 0.10%, 0.25% or 0.65%arginine or saline (control) and chow ad libitum for 12 days. Tumorgrowth in each arginine regimen animal was assessed for weight increasecompared to tumor growth exhibited in the saline (control) regimenanimals. Also, tumor growth in each animal was assessed for increase intumor weight compared to that animals pre-regimen tumor weight. In thismanner, each rat served as his or her own control in determining theeffect of the particular regimen or tumor growth. Urine ammonia levelswere also determined for each of the arginine regimen animals. Thesevalues were compared to the urine ammonia levels of saline (control)regimen animals.

TUMOR GROWTH DETERMINATIONS

Tumor growth was assessed by a measure of increase in tumor weightbefore and after the designated regimen as described in Example VIII.The results are demonstrated in FIG. 9. As demonstrated in FIG. 9,post-treatment tumor weight in the 0.10%, 0.25% and 0.65% arginineconcentration regimens did not vary significantly among groups. However,all 3 arginine regimen groups did exhibit post-treatment tumor weightssignificantly greater than the saline (control) regimen group tumorweights.

URINE AMMONIA DETERMINATIONS

Urine ammonia levels were assessed as a function of urine NH4 (mg./24hours) in each animal post-treatment. The levels obtained from animalsof each arginine treatment group were averaged and compared to theaverage level obtained in the saline (control) treated group. Asdemonstrated in FIG. 10, there was an inverse relationship between theconcentration of arginine given in the regimen to the level of urineammonia. Thus, the 0.25% arginine group exhibited higher urine ammonialevels compared to the 0.65% arginine group, and the 0.10% argininegroup exhibited higher urine ammonia levels compared to the 0.25%arginine group. All arginine groups (0.10%, 0.25%, 0.65%) displayedurine ammonia levels significantly higher than the saline (control)treated group.

                  TABLE X                                                         ______________________________________                                        Amino acid composition of                                                     parenteral regimens of rats                                                   Amino        Concentration of each amino                                      Acid         acid(mg) per 100 ml                                              Arg          0.1% arg   0.25% arg 0.65%                                       ______________________________________                                        Essential                                                                     LEUCINE      413        413       413                                         METHIONINE   413        413       413                                         PHENYLALANINE                                                                              413        413       413                                         LYSINE       300        300       300                                         VALINE       300        300       300                                         ISOLEUCINE   263        263       263                                         THREONINE    188        188       188                                         TRYPTOPHAN    94         94        94                                         HISTIDINE    206        206       206                                         Nonessential                                                                  ALANINE      1035       1035      1035                                        ARGININE     100        250       650                                         GLYCINE      1463       1203      515                                         PROLINE      340        340       340                                         SERINE       250        250       250                                         ______________________________________                                    

The TPN regimen was formulated with 500 ml of Aminess®, 500 ml D50W,electrolytes, and multivitamins. The amino acids, alanine, arginine,glycine, proline and serine were added to the TPN regimen. Thesesolutions were administered by continuous infusion through a centralvenous catheter, the flow rate being controlled with Holter 903 pump.The amino acid concentration(mg/100ml) in this table is theconcentration of the amino acids in the one liter TPN solution.

Control received i.v. 0.90% NaCl(saline) and chow ad libitum. The 0.10%arg group received a TPN formula with a final arginine concentration of0.1 g/100 ml. The 0.25% arg group received a TPN formula with a finalconcentration of 0.25 g/100 ml. The 0.65% arg group received a TPNformula with a final arginine concentration of 0.65 g/100 ml.

Those of skill in the art will recognize that, although the presentinvention is disclosed in terms of specific embodiments, one may departfrom such embodiments and still remain within the scope of theinvention. All such departures are considered to be within the scope ofpending claims.

What is claimed is:
 1. A formulation comprising a pharmacologicallyacceptable amount of:(a) a mixture of the following amino acids in aconcentration of between 0.01 to 5.0% by weight of theformulation:leucine; methionine; phenylalanine; lysine; valine;isoleucine; threonine; tryptophan; histidine; tyrosine; citrulline; andornithine (b) at least one non-essential amino acid in a concentrationof between 0.01-5% by weight of the formulation selected from the groupconsisting of:alanine; glycine; proline; and serine. the foregoing aminoacids being dispersed in a pharmacologically acceptable liquid medium.2. The formulation of claim 1 comprising a concentration of arginineless than about 0.10% by weight final concentration of the formulation.3. The formulation of claim 2 comprising a concentration of arginineless than about 0.05% by weight final concentration of the formulation.4. The method of claim 3, wherein administration of the formulation isparenteral.
 5. The formulation of claim 1, wherein citrulline andornithine are present in the following concentration ranges by weight inthe final feeding formulation:0.01-2.0% citrulline; and 0.01-0.5%ornithine.
 6. The formulation of claim 1, wherein the pharmacologicallyacceptable amount of essential amino acids comprises between about 0.01and about 5.0 grams of each essential amino acid for every 100milliliters of the formulation.
 7. The formulation of claim 1, whereinthe pharmacologically acceptable amount of the selected non-essentialamino acids comprise the following weight range of the amino acid forevery 100 milliliters of the formulation:200 to 3500 mg of alanine; 250to 2000 mg of glycine; 100 to 1500 mg of proline; 5 to 650 mg of serine;and 30 to 60 mg of tyrosine.
 8. The formulation of claim 1, wherein thepharmacologically acceptable amount of essential amino acids comprisethe following weight range of the amino acid for every 100 millilitersof the formulation:250 to 1400 mg of leucine; 200 to 1400 mg ofisoleucine; 200 to 1250 mg of valine; 100 to 900 mg of phenylalanine; 50to 850 mg of methionine; 150 to 750 mg of lysine; 85 to 500 mg ofhistidine; 100 to 550 mg of threonine; 50 to 200 mg of tryptophan. 9.The formulation of claim 1, wherein the pharmacologically acceptablemedium is a glucose solution.
 10. A method for inhibitingpolyamine-dependent malignant tumor growth in an animal with malignantdisease comprising administering to said animal the formulations definedin any one of claims 1, 2, 3, 5-9 and 12-13.
 11. A method of claim 10,wherein the animal is a human.
 12. The formulation of claim 1, whereinthe formulation is essentially arginine free.
 13. The formulation ofclaim 1, wherein the selected non-essential amino acid is alanine.
 14. Amethod for inhibiting Ward colon carcinoma tumor growth in an animalcomprising administering to the animal the formulations defined in anyone of claim 2, 3, 5-9, 12 or
 13. 15. The formulation of claim 14,wherein the concentration of alanine is in the weight range of between300 to 3,500 mg for every 100 milliliters of the formulation.